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Objective:To investigate changes in mRNA and protein expression of interleukin-27(IL-27)in glomerular podocyte injury caused by Puromycinonucleoside(PAN), and to explore the mechanism of the protective effect of Tacrolimus(FK506)on glomerular podocyte injury.Methods:Glomerular foot cells from mice were cultured in vitro and divided into 3 groups, which were the control group, PAN group and FK506 group.The morphology of 3 groups of foot cells was observed under a microscope after 8 h, 24 h, and 48 h treatment.The changes in IL-27 concentrations were detected by analyzing the enzyme-linked immunosorbent assay(ELISA)method.The cultured foot cells were then collected.The changes of IL-27 mRNA expression were measured by real-time fluorescence quantitative PCR(qPCR), and the changes of IL-27 protein expression were detected by Western blot. Results:(1)At each time point(8 h, 24 h, 48 h), the cells of the PAN group had smaller volume and different morphology than the cells of the control group, and the cells of the FK506 group was larger and fuller than the cells of the PAN group.(2)The concentrations of IL-27 in the PAN group [(110.00±3.52) ng/L, (302.00±6.23) ng/L, (397.00±8.92) ng/L] were significantly higher than those in the control group [(90.00±5.12) ng/L, (85.00±4.21) ng/L, (88.00±4.20) ng/L] and those in the FK506 group [(96.00±4.17) ng/L, (107.00±4.86) ng/L, (112.00±6.24) ng/L] at 8 h, 24 h and 48 h(all P<0.05). (3)At each time point(8 h, 24 h, 48 h), the IL-27 mRNA expression of the PAN group(1.25±0.11, 1.57±0.08, 1.73±0.13)was significantly higher than that of the control group(1.02±0.02, 1.10±0.04, 0.96±0.02)(all P<0.05). Compared with the control group, the IL-27 mRNA expression of the FK506 group did not significantly increase at 8 h (1.10±0.06), and showed a slight increase at 24 h and 48 h(1.21±0.04, 1.30±0.09). Compared with PAN group, HC506 group were all lower (all P<0.05). (4)At each time point(8 h, 24 h, 48 h), the expression of IL-27 protein in the PAN group(0.94±0.04, 1.56±0.07, 1.63±0.04) was significantly higher than that in the control group(0.83±0.04, 0.85±0.03, 0.83±0.05), there was significant difference(all P<0.05). Compared with the PAN group, the expression of IL-27 protein in the FK506 group(0.84±0.05, 0.89±0.04, 0.91±0.06)was not significantly different at 8 h, but decreased significantly at 24 h and 48 h, there was significant difference ( P<0.05). Conclusions:IL-27 is involved in the pathogenesis of kidney diseases.FK506 can prevent the glomerular podocyte injury by reducing the expression of IL-27.This study provides experimental basis for clinical application of FK506 in the treatment of kidney diseases.
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OBJECTIVE：To study the regulatory effects of stilbene glucosid e（TSG）on c-Jun N-terminal kinase （JNK）and protein phosphortase 2B（PP2B）in APP/PS1/Tau transgenic dementia （3×Tg-AD）mice，and to explore its potential mechanism of anti-Alzheimer’s disease （AD）. METHODS ：Totally 45 male 3×Tg-AD mice were randomly divided into model group ，positive control group （huperzine A ，0.15 mg/kg），TSG low-dose ，medium-dose and high-dose groups （0.033，0.1，0.3 g/kg），with 9 mice in each group. Another 9 normal male C 57BL/6J mice were included into normal control group. Administration groups were given relevant medicine intragastrically ，once a day ，for consecutive 60 d. Normal control group and model group were given constant volume of normal saline intragastrically. After medication ，Morris water maze experiment was used to test the spatial learning and memory ability of mice in each group ；Nissl staining was used to observe the changes of Nissl bodies in cerebral cortex and hippocampus ；mRNA and protein expressions of JNK and PP 2B were detected by qRT-PCR and Western blotting assay. RESULTS：Compared with normal control group ，the escape latency was significantly prolonged （P＜0.01），the retention time of the original platform quadrant was significantly shortened （P＜ and the times of crossing the platform was significantly reduced in model group （P＜0.01）；the number of Nissl bodies in cerebral cortex and hippocampus was significantly 729011126@qq.com reduced，the staining was slight ；the relative expressions of JNK mRNA and protein were significantly increased （P＜ 0.01），and the relative expressi ons of PP 2B mRNA and protein were significantly decreased （P＜0.01）. Compared with model group ，the escape latency was significantly shortened in positive control group and TSG groups （P＜0.01）；the retention time of the original platform quadrant was significantly prolonged （P＜0.01）；the times of crossing the platform was significantly increased （P＜0.01）；the number of Nissl bodies in cerebral cortex and hippocampus was increased significantly ，the staining was heavy ；the relative expression of JNK protein was significantly decreased（P＜0.05 or P＜0.01），the relative expressions of PP 2B mRNA and protein were significantly increased （P＜0.01）， while the relative expression of JNK mRNA was significantly decreased in TSG high-dose group （P＜0.05）. CONCLUSIONS ：TSG can improve the learning and memory ability and neuronal damage of 3 × Tg-AD mice. The mechanism may be related to down-regulating the transcription and expression of protein kinase JNK ，up-regulating the transcription and expression of protein phosphatase PP 2B.
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OBJECTIVE： To observe the effects of stilbene glycosidec （TSG） on okadaic acid （OA）-induced Tau protein phosphorylation in NG108-15 cells， and to investigate the potential anti-Alzheimer’s disease （AD） mechanism of this compound. METHODS： AD model of NG108-15 cells was induced by OA. The survival rate of NG108-15 cells was observed by MTT assay after pretreated with low-dose， medium-dose and high-dose of TSG （50， 100， 200 μmol/L）. The apoptosis of NG108-15 cells was detected by AO/EB double fluorescence staining. The protein and mRNA expression of CDK5 and GSK3β， and the protein expression of Tau and p-Tau were detected by Western blotting assay and RT-PCR. The distribution of CDK5， GSK3β and Tau protein were detected by immunofluorescence. RESULTS： The normal morphology of NG108-15 cells was observed in normal control group， but CDK5， GSK3β and Tau protein were not found or few was found. Contracted or globular early apoptotic cells were observed in model gorup； the distribution of CDK5， GSK3β and Tau protein was increased， while survival rate of the cells was decreased； protein and mRNA expression of CDK5 and GSK3β as well as ratio of the relative expression of p-Tau to that of Tau （p-Tau/Tau） were all increased significantly （P＜0.05 or P＜0.01）. After pretreatment of TSG， the distribution of early apoptotic cells as well as CDK5， GSK3β and Tau protein were all decreased to some extent in administration groups， while survival rates of the cells were increased significantly. Protein expression of CDK5 and p-Tau/Tau in medium-dose group and high-dose group as well as mRNA expression of CDK5， protein and mRNA expression of GSK3β in administration group were decreased significantly （P＜0.05）. CONCLUSIONS： TSG can protect against AD model cells， the effects of which may be associated with improving survival rate of the cells， down-regulating the protein expression and gene transcription level of phosphokinase CDK5 and GSK3β， inhibiting Tau protein phosphorylation.
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OBJECTIVE:To isolate and purify three principal degraded impurities of mitiglinide calcium (impurity A,B,C) and identify their structures,establish HPLC method for content determination of impurity A,B,C. METHODS:Mitiglinide calci-um was used as raw material and reacted with acid;3 impurities were then separated by HPLC and their structures were elucidated by IR,MS,1H NMR,13C NMR,LC-ESI-MS and ORD. 3 impurities of 3 batches of mitiglinide calcium were determined,and the determination was performed on Agilent Extend-C18 column with mobile phase consisted of 0.01 mol/L sodium acetate solution-ace-tonitrile-triethylamine(60:40:0.1,pH=3.0)at the flow rate of 1.0 ml/min. The detection wavelength was set at 210 nm and sam-ple size was 20 μl. The response tests of 3 impurities and mitiglinide calcium were conducted. RESULTS:After treated with acid, impurity A,B,C had been obtained,and their purity were 99.05%,98.87%,99.98%,respectively after isolation and purifica-tion;after identifying the structure, 3 impurities were S-2-bezylsuccinic acid, S-2-bezylsuccinic acid-4-methyl ester, methyl (2S)-2-benzyl-3-(cis-hexahydroisoindolin-2-ylcarbonyl) propionate;methodological study of content determination of impurities were all up to the requirement. The linear range of impurity A,B,C were 0.387 5-3.875,0.395-3.95 and 0.392 5-3.925 μg/ml(all r were 1.000 0). The response value of impurity A,B,C and mitiglinide calcium were 2.316 1,2.636 1,2.617 8 and 2.620 4,re-spectively. CONCLUSIONS:The structures of 3 principal degraded impurities of mitiglinide calcium have been identified and con-firmed;the content of them can be determined by HPLC main component self-comparison method.
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Objective To evaluate the treatment of current status and prognosis in childhood APL with APL2008 ,which was administrated since 2008 in our center .Methods A total of 43 children with newly diagnosed APL between 2008 to 2014 were studied retrospectively .Treatment options and current status were summarized from 28 patients who received APL2008 therapy . Results Studied 43 patients were at median age of 8 years and 4 months ,with 28 boys and 15 girls .The main clinical manifestations were infection ,anemia ,bleeding ,fever ,hepatomegaly ,splenomegaly and lymphadenopathy .The proportions of low ,intermediate and high risk groups were 27 .9% ,48 .8% and 23 .3% ,respectively .Eleven cases could be diagnosed as DIC .Bone marrow morphology showed abnormal elevation of promyelocyte .37 patients had distinctive immunophenotype such as frequent expression of CD33 , CD117 and MPO .PML/RARαfusion gene positive rate was 100% in 43 children and cytogenetic analysis were positive in 37 cases , of which specific genetic lesion in APL cells with t (15 ;17)(q22 ;q12) was found in 28 cases ,and karyotypes was found in 9 cases as infrequent chromosomal abnormalities .In 43 patients ,4 cases were early dead from intracranial hemorrhage at early stage ,and 11 cases were given up early .There were only 2 cases dead ,2 cases relapsed and 1 case lost among 28 APL children ,which enabled ef‐ficacy analysis possible .96 .4% of these 28 cases achieved HCR .The 2 year Kaplan Meier estimates of OS and EFS were 85 .9% ± 7 .6% and 80 .4% ± 8 .8% .But OS and EFS would be 94 .7% ± 5 .1% and 88 .9% ± 7 .4% if 3 patients who had non standard treat‐ment were excluded .Conclusion Childhood APL were characterized by anemia ,bleeding ,fever and infiltration .APL′s coincidence rate between PML/RARa fusion gene and morphology ,immunology and cytogenetics were 95 .3% ,90 .2% and 86 .5% ,respective‐ly .APL2008 significantly improved the prognosis of APL .
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Objective To screen the sensing elements for TNT detection in Escherichia coli genome.Methods A genome promoter library with cutting E.coli K-12 MG1655 genome was constructed.Bacterial luciferase luxCDABE was used as a reporter gene during promoter screening.We discovered TNT sensing elements through several rounds of screen-ing.Through analysis of sensitivity, specificity and timeliness, the promoter activity of the elements was evaluated,and the functional sequence of the elements was further confirmed.Results and Conclusion We successfully constructed an E.co-li K-12 MG1655 genome library , from which a TNT sensing element was discovered,which had a good performance in the analysis of sensitivity, specificity and timeliness.In this study, we reported that the topAp4 is a TNT sensing element for the first time.We also verified its excellent promoter activity.
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Objective:To design and construct the replication-deficient recombinant adenovirus Ad-siCTGF which can silence the expression of connective tissue growth factor (CTGF) by RNA interference and verified its function. Methods:A specific sequence, which was verified to be able to silence CTGF gene with high efficiency, was cloned into pSES-HUS vector to produce the shuttle plasmid pSES-siCTGF. The plasmid after Pme Ⅰ linearization was cotransduced with pAdEasy into BJ5183 E.coli strains to construct recombinant vector Ad-siCTGF. After linearization treatment with Pac Ⅰ enzyme digestion Ad-siCTGF was transfected into HEK293 cells via liposome mediation. The recombinant adenovirus was packaged. The titer of the Ad-siCTGF was increased after three times of cross-infection. 4T1 cells were infected with the adenovirus. The silencing efficiency was tested by real-time fluorescence quantitative (RFQ)-PCR and Western blotting.Results:Pac Ⅰ enzyme digestion electrophoresis indentified that recombinant adenovirus was successfully constructed. The titer of the recombinant adenovirus Ad-siCTGF was 2.6×10~(10) pfu/mL after amplification and purification. The CTGF mRNA and protein expression in 4T1 cells were decreased by 36.27% and 31.56%, respectively, compared with the control groups.Conclusion:The recombinant adenovirus which can silence the expression of CTGF was successfully constructed. It laid a good foundation for further investigation of the action mechanism of CTGF in tumor cells.
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To identify the structure of three related substances in potassium sodium dehydroandrographolide succinate (PSDS), an HPLC preparation method was used to separate the impurities. These main impurities were identified using LC-ESI/TOFMS, LC-ESI/MSn, NMR, UV and IR. One of the main impurities was a hydrolyzed and oxidized product of PSDS, which has not been reported previouely. The other two impurities were hydrolyzed products of PSDS after losing different succinic acids. The results indicate that PSDS can be easily hydrolyzed and oxidized. It should be stored at cool and dry places.
